Podophyllotoxin derivatives and their use

ABSTRACT

Provided herein are compounds, compositions, and methods for treating cancer in a subject in need thereof.

FIELD

The present disclosure relates to derivatives of podophyllotoxin,compositions thereof, and using them for treating various types ofcancer in a subject and/or delaying or regressing various types of tumorgrowths in a subject.

STATE OF THE ART

Podophyllotoxin is a known compound having the formula:

The compound shows activity as an antiviral and as an antineoplasticagent. This disclosure relates to derivatives of podophyllotoxin thatare useful for treating cancer.

SUMMARY

In one aspect, provided herein is a compound of formula (I):

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing, wherein

R¹ is H or has a structure of:

R³ is H, an optionally substituted C₁-C₈ alkyl, an optionallysubstituted 3-10 membered cycloalkyl, an optionally substituted 3-10membered heterocyclyl, an optionally substituted 6-10 membered aryl, oran optionally substituted 5-10 membered heteroaryl;

R⁵ is H or has a structure of:

and

R¹¹ and R¹² independently is an optionally substituted C₁-C₈ alkyl.

In another aspect, provided herein is a pharmaceutical compositioncomprising a compound provided herein, and at least one pharmaceuticallyacceptable excipient.

In another aspect, provided herein is a method of treating cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a compound provided herein.

In another aspect, provided herein is a method of delaying or regressingtumor growth in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of a compound providedherein.

In another aspect, provided herein is a method of treating cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositionprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the ¹H Nuclear Magnetic Resonance spectra of N2. FIGS. 1Band 1F show the ¹H Nuclear Magnetic Resonance spectra of TargetCompounds 1 and 4 respectively. FIGS. 1C-1E show the ¹H Nuclear MagneticResonance spectra of Target Compound 3.

FIG. 2 shows the ¹³C Nuclear Magnetic Resonance spectrum of N2.

FIGS. 3A-3D show the Liquid Chromatography-Mass Spectra of N2 and TargetCompounds 1, 3, and 4 respectively.

FIG. 4A shows the antitumor activity of formulated N2-HCl (i.v.) intreatment of COLO205 Human Colon Cancer Xenograft Model. FIG. 4B showsthe antitumor activity of N2-HCl (i.p.) in treatment of COLO205 HumanColon Cancer Xenograft Model.

FIG. 5A shows the antitumor activity of formulated N2 (i.v.) intreatment of HCT116 Human Colon Cancer Xenograft Model. FIG. 5B showsthe antitumor activity of N2-HCl (i.p.) in treatment of HCT116 HumanColon Cancer Xenograft Model.

FIG. 6 shows the tumor growth curves of the different groups of micedescribed in Table 1.7.

FIG. 7 shows the antitumor activity of formulated N2 and Imatinib in thetreatment of K562 Xenograft Model.

DETAILED DESCRIPTION

The compounds of the disclosure are active against tumors in mice andare generally well tolerated or are contemplated to be so. They areuseful for treating various types of cancer and can be formulated toprepare pharmaceutical preparations, e.g. for oral, topical, orparenteral administration.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations. Each numerical parameter should atleast be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques.

Throughout this disclosure, the text refers to various embodiments ofthe present compounds, compositions, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather, it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anexcipient” includes a plurality of excipients.

As used herein, the following definitions shall apply unless otherwiseindicated. Further, if any term or symbol used herein is not defined asset forth below, it shall have its ordinary meaning in the art.

“Comprising” is intended to mean that the compositions and methodsinclude the recited elements, but not excluding others. “Consistingessentially of” when used to define compositions and methods, shall meanexcluding other elements of any essential significance to thecombination. For example, a composition consisting essentially of theelements as defined herein would not exclude other elements that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. “Consisting of” shall mean excluding more than trace amountof other ingredients and substantial method steps recited. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

The term “alkyl” refers to a monovalent, saturated aliphatic hydrocarbonradical having the indicated number of carbon atoms. For example, a “C1-6 alkyl” or an “alkyl of 1-6 carbons” or “Alk 1-6” would refer to anyalkyl group containing one to six carbons in the structure. “C 1-20alkyl” refers to any alkyl group having one to twenty carbons. Alkyl maybe a straight chain (i.e. linear) or a branched chain. Representativeexamples lower alkyl radicals include methyl, ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, sec-butyl,tert-butyl, tert-pentyl, n-heptyl, n-octyl, and the like, along withbranched variations thereof. The radical may be optionally substitutedwith substituents at positions that do not significantly interfere withthe preparation of compounds falling within the scope of this inventionand that do not significantly reduce the efficacy of the compounds. Thealkyl may be optionally substituted with one to five substituentsindependently selected from the group consisting of halo, lower alkoxy,hydroxy, cyano, nitro, or amino.

The term “alkoxy” refers to a monovalent radical of the formula RO—,where R is an alkyl as defined herein. Lower alkoxy refers to an alkoxyof 1-6 carbon atoms, with higher alkoxy is an alkoxy of seven or morecarbon atoms. Representative lower alkoxy radicals include methoxy,ethoxy, n-propoxy, n-butoxy, n-pentyloxy, n-hexyloxy, isopropoxy,isobutoxy, isopentyloxy, amyloxy, sec-butoxy, tert-butoxy,tert-pentyloxy, and the like. Higher alkoxy radicals include thosecorresponding to the higher alkyl radicals set forth herein. The radicalmay be optionally substituted with substituents at positions that do notsignificantly interfere with the preparation of compounds falling withinthe scope of this invention and that do not significantly reduce theefficacy of the compounds. The radical may be optionally substitutedwith one to five substituents independently selected from the groupconsisting of halo, lower alkyl, lower alkoxy, hydroxy, cyano, nitro, oramino.

The term “cycloalkyl” refers to a monovalent, alicyclic, saturatedhydrocarbon radical having three or more carbons forming the ring. Whileknown cycloalkyl compounds may have up to 30 or more carbon atoms,generally there will be three to seven carbons in the ring. The latterinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cycloheptyl. The radical may be optionally substituted withsubstituents at positions that do not significantly interfere with thepreparation of compounds falling within the scope of this invention andthat do not significantly reduce the efficacy of the compounds. Thecycloalkyl is optionally substituted with one to five substituentsindependently selected from the group consisting of halo, lower alkyl,lower alkoxy, hydroxy, cyano, nitro, amino, halogenated lower alkyl,halogenated lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl, loweralkylcarbonyloxy, and lower alkylcarbonylamino.

The term “hydroxycarbonyl” is a monovalent radical having the formula—C(O)OH.

The term “lower alkoxycarbonyl” is a monovalent radical having theformula —C(O)OAlk, where Alk is lower alkyl.

The term “lower alkylcarboxyloxy” is a monovalent radical having theformula —OC(O)Alk, where Alk is lower alkyl.

The term “lower alkylcarbonylamino” is a monovalent radical having theformula —NHC(O)Alk, where Alk is lower alkyl.

The term “alkylamino” is a monovalent radical having the formula —NR₁,R₂ where R₁ is alkyl and R₂ is hydrogen or alkyl and the alkyl isoptionally substituted.

“Amino” refers to the group —NH₂.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl (Ph)) ormultiple condensed rings (e.g., naphthyl or anthryl) which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with 1 to5, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, guanidino,substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are defined herein.

A “halo” substituent is a monovalent halogen radical chosen from chloro,bromo, iodo, and fluoro. A “halogenated” compound is one substitutedwith one or more halo substituent.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Preferred heteroaryls include 5or 6 membered heteroaryls such as pyridinyl, pyrrolyl, indolyl,thiophenyl, and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or partially saturated, but not aromatic, grouphaving from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatomsselected from the group consisting of nitrogen, sulfur, or oxygen. C_(x)cycloalkyl refers to a heterocycloalkyl group having x number of ringatoms including the ring heteroatoms. Heterocycle encompasses singlering or multiple condensed rings, including fused bridged and spiro ringsystems. In fused ring systems, one or more the rings can be cycloalkyl,aryl or heteroaryl provided that the point of attachment is through thenon-aromatic ring. In one embodiment, the nitrogen and/or sulfur atom(s)of the heterocyclic group are optionally oxidized to provide for theN-oxide, sulfinyl, sulfonyl moieties.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 5 or preferably 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

Examples of heterocycle and heteroaryl include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

A “cyclic amino” is a monovalent radical of a saturated 5-, 6-, or7-membered cyclic amine ring having no more than one additional heteroatom such as nitrogen, oxygen, or sulfur. Representative examplesinclude, e.g., 1-pyrrolidino, 1-piperidino, morpholino, piperazino,3-benzylpiperidino, and the like. These may be substituted orunsubstituted. If substituted, generally they will have no more than 2substituents chosen from lower alkyl, lower cycloalkyl, hydroxy loweralkyl, phenyl (substituted or unsubstituted), benzyl (substituted orunsubstituted), aminocarbonylmethyl, lower alkylaminocarbonylmethyl,amino, mono- or di-lower alkylamino, cyclic amino, or a 5- or 6-memberedheterocyclic ring.

Other chemical terms are given their standard meaning as understood byone of skill in the art with guidance from standard texts anddictionaries.

The term “MTD” is the abbreviation for maximum tolerated does.

The term “nM” is the abbreviation for nanomolar.

The term “ip” is the abbreviation for intraperitoneal.

“Treating” or “treatment” of a disease in a patient refers to 1)preventing the disease from occurring in a patient that is predisposedor does not yet display symptoms of the disease; 2) inhibiting thedisease or arresting its development; or 3) ameliorating or causingregression of the disease

“Subject” refers to mammals and includes humans and non-human mammals.Examples of patients include, but are not limited to mice, rats,hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, sheep, cows,and humans. In some embodiments, patient refers to a human.

The term “pharmaceutically acceptable” refers to safe and non-toxic forin vivo, preferably, for human administration.

“Pharmaceutically acceptable salt” refers to a salt that ispharmaceutically acceptable. Any compound described herein may beadministered as a pharmaceutically acceptable salt.

“Salt” refers to an ionic compound formed between an acid and a base.When the compound provided herein contains an acidic functionality, suchsalts include, without limitation, alkali metal, alkaline earth metal,and ammonium salts. As used herein, ammonium salts include, saltscontaining protonated nitrogen bases and alkylated nitrogen bases.Exemplary and non-limiting cations useful in pharmaceutically acceptablesalts include Na, K, Rb, Cs, NH₄, Ca, Ba, imidazolium, and ammoniumcations based on naturally occurring amino acids. When the compoundsutilized herein contain basic functionality, such salts include, withoutlimitation, salts of organic acids, such as carboxylic acids andsulfonic acids, and mineral acids, such as hydrogen halides, sulfuricacid, phosphoric acid, and the likes. Exemplary and non-limiting anionsuseful in pharmaceutically acceptable salts include oxalate, maleate,acetate, propionate, succinate, tartrate, chloride, sulfate, bisulfate,mono-, di-, and tribasic phosphate, mesylate, tosylate, and the likes.

“Effective amount” or dose of a compound or a composition, refers tothat amount of the compound or the composition that results in anintended result as desired based on the disclosure herein. Effectiveamounts can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., and without limitation, bydetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio LD₅₀/ED₅₀.

“Therapeutically effective amount” or dose of a compound or acomposition refers to that amount of the compound or the compositionthat results in reduction or inhibition of symptoms or a prolongation ofsurvival in a patient. The results may require multiple doses of thecompound or the composition.

The terms “optional” or “optionally” as used throughout thespecification means that the subsequently described event orcircumstance may but need not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. For example, “the nitrogen atom is optionally oxidized toprovide for the N-oxide (N→O) moiety” means that the nitrogen atom maybut need not be oxidized, and the description includes situations wherethe nitrogen atom is not oxidized and situations where the nitrogen atomis oxidized.

The term “optionally substituted” refers to a substituted orunsubstituted group. The group may be substituted with one or moresubstituents, such as e.g., 1, 2, 3, 4 or 5 substituents. Preferably,the substituents are selected from the functional groups providedherein. In certain more preferred embodiments, the substituents areselected from oxo, halo, —CN, NO₂, —CO₂R¹⁰⁰, —OR¹⁰⁰, —SR¹⁰⁰, —SOR¹⁰⁰,—SO₂R¹⁰⁰, —NR¹⁰¹R¹⁰², —CONR¹⁰¹R¹⁰², —SO₂NR¹⁰¹R¹⁰², C₁-C₆ alkyl, C₁-C₆alkoxy, —CR¹⁰⁰═C(R¹⁰⁰)₂, —CCR¹⁰⁰, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocyclyl, C₆-C₁₂ aryl and C₅-C₁₂ heteroaryl, wherein each R¹⁰⁰independently is hydrogen or C₁-C₈ alkyl; C₃-C₁₂ cycloalkyl; C₃-C₁₀heterocyclyl; C₆-C₁₂ aryl; or C₂-C₁₂ heteroaryl; wherein each alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substitutedwith 1-3 halo, 1-3 C₁-C₆ alkyl, 1-3 C₁-C₆ haloalkyl or 1-3 C₁-C₆ alkoxygroups. More preferably, the substituents are selected from the groupconsisting of chloro, fluoro, —OCH₃, methyl, ethyl, iso-propyl,cyclopropyl, —OCF₃, —CF₃ and —OCHF₂.

R¹⁰¹ and R¹⁰² independently are hydrogen; C₁-C₈ alkyl, optionallysubstituted with —CO₂H or an ester thereof, C₁-C₆ alkoxy, oxo,—CR¹⁰³═C(R¹⁰³)₂, —CCR, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₆-C₁₂aryl, or C₂-C₁₂ heteroaryl, wherein each R¹⁰³ independently is hydrogenor C₁-C₈ alkyl; C₃-C₁₂ cycloalkyl; C₃-C₁₀ heterocyclyl; C₆-C₁₂ aryl; orC₂-C₁₂ heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with 1-3 alkyl groups or 1-3 halogroups, or R¹⁰¹ and R¹⁰² together with the nitrogen atom they areattached to form a 5-7 membered heterocycle.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“alkoxycarbonylalkyl” refers to the group (alkoxy)-C(O)-(alkyl)-.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

Compounds

In one aspect, provided herein is a compound of formula (I):

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing, wherein

R¹ is H or has a structure of:

R³ is H, an optionally substituted C₁-C₈ alkyl, an optionallysubstituted 3-10 membered cycloalkyl, an optionally substituted 3-10membered heterocyclyl, an optionally substituted 6-10 membered aryl, oran optionally substituted 5-10 membered heteroaryl;

R⁵ is H or has a structure of:

and

R¹¹ and R¹² independently is an optionally substituted C₁-C₈ alkyl.

In one embodiment, R¹ is H. In another embodiment, R¹ has a structureof:

In one embodiment, R⁵ is H. In another embodiment, R⁵ is:

In one embodiment, R¹¹ is an optionally substituted C₁-C₈ alkyl. Inanother embodiment, R¹¹ is C₁-C₈ alkyl. In another embodiment, R¹¹ ismethyl.

In one embodiment, R¹² is an optionally substituted C₁-C₈ alkyl. Inanother embodiment, R¹² is C₁-C₈ alkyl. In another embodiment, R¹² ismethyl.

In one embodiment, R³ is H. In one embodiment, R³ is an optionallysubstituted C₁-C₈ alkyl. In another embodiment, R³ is C₁-C₈ alkyl. Inanother embodiment, R³ is an optionally substituted 3-10 memberedcycloalkyl. In another embodiment, R³ is an optionally substituted 3-10membered heterocyclyl. In another embodiment, R³ is an optionallysubstituted 6-10 membered aryl. In another embodiment, R³ is anoptionally substituted 5-10 membered heteroaryl. In another embodiment,R³ is ethyl.

In one embodiment, the compound of formula (I) has the structure of:

wherein the variables are defined as above.

In one embodiment, the compound of formula (I) is:

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing.

In one embodiment, the compound of formula (I) is:

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing.

In one embodiment, the compound of formula (I) is:

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing.

In one embodiment, the compound of formula (I) is:

-   -   or        or an N-oxide thereof, or a pharmaceutically acceptable salt of        each thereof, or a pharmaceutically acceptable solvate of each        of the foregoing.

In one embodiment, provided herein are individual stereoisomers, such asenantiomers, of the compounds provided herein. In one embodiment, thecompounds provided herein have a stereochemical configuration as shownbelow:

-   -   or

Podophyllotoxin derivatives can be prepared by the skilled artisan basedon methods described herein and/or in U.S. Pat. No. 8,158,809, oradapting those methods.

Pharmaceutical Composition

In another aspect, provided herein is a pharmaceutical compositioncomprising a compound provided herein, and at least one pharmaceuticallyacceptable excipient.

In one embodiment, the pharmaceutical composition is a solid form,liquid form, injectable form, or a liposomal formulation. In anotherembodiment, the pharmaceutical composition is a solid form. In anotherembodiment, the pharmaceutical composition is a liquid form. In anotherembodiment, the pharmaceutical composition is an injectable form. Inanother embodiment, the pharmaceutical composition is a liposomalformulation. In another embodiment, the pharmaceutical composition is amicelle formulation.

This aspect of the disclosure is a pharmaceutical composition useful fortreating cancer in a warm-blooded animal, which composition comprisescompound of the disclosure as defined herein in combination with apharmaceutically acceptable excipient. The composition is prepared inaccordance with known formulation techniques to provide a compositionsuitable for oral, topical, transdermal, rectal, by inhalation,parenteral (intravenous, intramuscular, or intraperitoneal)administration, and the like. Detailed guidance for preparingcompositions of the invention are found by reference to the 18^(th) or19^(th) Edition of Remington's Pharmaceutical. Sciences, Published bythe Mack Publishing Co., Easton, Pa. 18040. The pertinent portions areincorporated herein by reference.

Unit doses or multiple dose forms are contemplated, each offeringadvantages in certain clinical settings. The unit dose would contain apredetermined quantity of active compound calculated to produce thedesired effect(s) in the setting of treating cancer. The multiple doseform may be particularly useful when multiples of single doses, orfractional doses, are required to achieve the desired ends. Either ofthese dosing forms may have specifications that are dictated by ordirectly dependent upon the unique characteristic of the particularcompound, the particular therapeutic effect to be achieved, and anylimitations inherent in the art of preparing the particular compound fortreatment of cancer.

A unit dose will contain a therapeutically effective amount sufficientto treat cancer in a subject and may contain from about 1.0 to 1000 mgof compound, for example about 50 to 500 mg.

The compound will preferably be administered orally in a suitableformulation as an ingestible tablet, a buccal tablet, capsule, caplet,elixir, suspension, syrup, trouche, wafer, lozenge, and the like.Generally, the most straightforward formulation is a tablet or capsule(individually or collectively designated as an “oral dosage unit”).Suitable formulations are prepared in accordance with a standardformulating techniques available that match the characteristics of thecompound to the excipients available for formulating an appropriatecomposition. A tablet or capsule will preferably contain about 50 toabout 500 mg of a compound of Formula (I).

The form may deliver a compound rapidly or may be a sustained-releasepreparation. The compound may be enclosed in a hard or soft capsule, maybe compressed into tablets, or may be incorporated with beverages, foodor otherwise into the diet. The percentage of the final composition andthe preparations may, of course, be varied and may conveniently rangebetween 1 and 90% of the weight of the final form, e.g., tablet. Theamount in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions according tothe current invention are prepared so that an oral dosage unit formcontains between about 5.0 to about 50% by weight (% w) in dosage unitsweighing between 5 and 1000 mg.

The suitable formulation of an oral dosage unit may also contain: abinder, such as gum tragacanth, acacia, corn starch, gelatin; sweeteningagents such as lactose or sucrose; disintegrating agents such as cornstarch, alginic acid and the like; a lubricant such as magnesiumstearate; or flavoring such a peppermint, oil of wintergreen or thelike. Various other material may be present as coating or to otherwisemodify the physical form of the oral dosage unit. The oral dosage unitmay be coated with shellac, a sugar or both. Syrup or elixir may containthe compound, sucrose as a sweetening agent, methyl and propylparabensas a preservative, a dye and flavoring. Any material utilized should bepharmaceutically-acceptable and substantially non-toxic. Details of thetypes of excipients useful may be found in the nineteenth edition of“Remington: The Science and Practice of Pharmacy,” Mack PrintingCompany, Easton, Pa. See particularly chapters 91-93 for a fullerdiscussion.

A compound may be administered parenterally, e.g., intravenously,intramuscularly, intravenously, subcutaneously, or intraperitoneally.The carrier or excipient or excipient mixture can be a solvent or adispersive medium containing, for example, various polar or non-polarsolvents, suitable mixtures thereof, or oils. As used herein “carrier”or “excipient” means a pharmaceutically acceptable carrier or excipientand includes any and all solvents, dispersive agents or media,coating(s), antimicrobial agents, iso/hypo/hypertonic agents,absorption-modifying agents, and the like. The use of such substancesand the agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use in therapeutic compositionsis contemplated. Moreover, other or supplementary active ingredients canalso be incorporated into the final composition.

Solutions of the compound may be prepared in suitable diluents such aswater, ethanol, glycerol, liquid polyethylene glycol(s), various oils,and/or mixtures thereof, and others known to those skilled in the art.

The pharmaceutical forms suitable for injectable use include sterilesolutions, dispersions, emulsions, and sterile powders. The final formmust be stable under conditions of manufacture and storage. Furthermore,the final pharmaceutical form must be protected against contaminationand must, therefore, be able to inhibit the growth of microorganismssuch as bacteria or fungi. A single intravenous or intraperitoneal dosecan be administered. Alternatively, a slow long term infusion ormultiple short term daily infusions may be utilized, typically lastingfrom 1 to 8 days. Alternate day or dosing once every several days mayalso be utilized.

Sterile, injectable solutions are prepared by incorporating a compoundin the required amount into one or more appropriate solvents to whichother ingredients, listed above or known to those skilled in the art,may be added as required. Sterile injectable solutions are prepared byincorporating the compound in the required amount in the appropriatesolvent with various other ingredients as required. Sterilizingprocedures, such as filtration, then follow. Typically, dispersions aremade by incorporating the compound into a sterile vehicle which alsocontains the dispersion medium and the required other ingredients asindicated above. In the case of a sterile powder, the preferred methodsinclude vacuum drying or freeze drying to which any required ingredientsare added.

In one embodiment, the compositions provided here are for injection,such as solutions for injection. In one embodiment, the compoundsprovided herein can be administered as an injectable solution. In oneembodiment, the injectable solution comprises at least onepharmaceutically acceptable excipient. In one embodiment, thepharmaceutically acceptable excipient is one or more of D-mannitol,L-lactic acid, a pH adjusting agent, and sterile water. A non-limitingexample of a pH adjusting agent is sodium hydroxide. In one embodiment,Compound 801.HCl is administered as an injectable solution (see Table Abelow).

TABLE A Formulated Bulk Solution: (20 L/20.00 kg) Final Product Fill:5.00 ± 0.10 g Reference to Amount per Component Quality batch Amount pervial Concentration 801•HCl Formulate^(a) Meets 697.7 g 30.0 mg (freebase) 6 mg/mL (free base) specification (formulate) 174.4 mg (formulate)34.9 mg (formulate) d-Mannitol USP 168.0 g 42.0 mg 8.4 mg/mL L-Lacticacid USP 18.02 g 4.50 mg 0.901 mg/mL Sodium hydroxide NF As needed to Asneeded As needed adjust pH to 3.5 Sterile water for USP QS to 20.00 kgQS to 5.00 mL QS to 1 mL injection ^(a)801•HCl Formulate = 801 freebase•HCl•2 MPEG-2000-DSPE QS = quantity sufficient

In all cases the final form, as noted, must be sterile and must also beable to pass readily through an injection device such as a hollowneedle. The proper viscosity may be achieved and maintained by theproper choice of solvents or excipients. Moreover, the use of molecularor particulate coatings such as lecithin, the proper selection ofparticle size in dispersions, or the use of materials with surfactantproperties may be utilized.

Prevention or inhibition of growth of microorganisms may be achievedthrough the addition of one or more antimicrobial agents such aschlorobutanol, ascorbic acid, parabens, thermerosal, or the like. It mayalso be preferable to include agents that alter the tonicity such assugars or salts.

Although the compounds of this disclosure tend to be water soluble, insome cases, e.g., where a compound of the invention is less watersoluble, it may be useful to provide liposomal delivery. The systemrestrains the compound of the invention by incorporating, encapsulating,surrounding, or entrapping the compound of the invention in, on, or bylipid vesicles or liposomes, or by micelles.

Liposomes have been used successfully to administer medications tocancer patients, and have been shown to be useful clinically in thedelivery of anticancer drugs such as doxorubicin, daunorubicin, andcisplatinum complexes. Forssen, et al., Cancer Res. 1992, 52: 3255-3261;Perex-Soler, et al., Cancer Res. 1990, 50: 4260-4266; and, Khokhar, etal., J. Med. Chem. 1991, 34: 325-329, all of which are incorporatedherein in their entireties by reference.

Similarly, micelles have also been used to deliver medications topatients, (Broden et al., Acta Pharm Suec. 19: 267-284 (1982)) andmicelles have been used as drug carriers and for targeted drug delivery,(D. D. Lasic, Nature 335: 279-280 (1992); and, Supersaxo et al., PharmRes. 8: 1280-1291 (1991)), including cancer medications, (Fung et al.,Biomater. Artif Cells. Artif Organs 16: 439 et seq. (1988); and Yokoyamaet al., Cancer Res. 51: 3229-3236 (1991)), all of which are incorporatedherein in their entireties by reference.

The liposomes and/or micelles containing the compound of the inventioncan be administered to a cancer patient, typically intravenously.Further guidance for preparing liposomal compositions useful in thisinvention may be found in U.S. Pat. No. 6,096,336, which is incorporatedherein by reference.

Methods of Treatment

In another aspect, provided herein is a method of treating cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a compound provided herein.

In one embodiment, the therapeutically effective amount of the compoundadministered is about 15 mg/m² to about 1500 mg/m². In anotherembodiment, the therapeutically effective amount of a compound thecompound administered is about 50 mg/m² to about 1000 mg/m². In anotherembodiment, the therapeutically effective amount of the compoundadministered is about 100 mg/m² to about 750 mg/m². In anotherembodiment, the therapeutically effective amount of the compoundadministered is about 250 mg/m² to about 500 mg/m².

In one embodiment, the cancer is colon cancer, non-small lung cancer,chronic myeloid leukemia, acute myeloid leukemia, small cell lungcancer, cervical cancer, stomach cancer, breast cancer, prostate cancer,skin cancer, melanoma, lymphoma, brain tumor, sarcoma, testicle cancer,ovarian cancer, renal cancer, head and neck cancer, liver cancer,leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia,hairy cell leukemia, T-cell prolymphocytic leukemia, large granularlymphocytic leukemia, adult T-cell leukemia, and a blood cancer.

In one embodiment, the cancer comprises a solid tumor.

In another aspect, provided herein is a method of delaying or regressingtumor growth in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of a compound providedherein.

In one embodiment, the tumor growth is a solid tumor.

In one embodiment, the cancer is colon cancer, non-small lung cancer,chronic myeloid leukemia, acute myeloid leukemia, small cell lungcancer, cervical cancer, stomach cancer, breast cancer, prostate cancer,skin cancer, melanoma, lymphoma, brain tumor, sarcoma, testicle cancer,ovarian cancer, renal cancer, head and neck cancer, liver cancer,leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia,hairy cell leukemia, T-cell prolymphocytic leukemia, large granularlymphocytic leukemia, adult T-cell leukemia, and a blood cancer.

In another aspect, provided herein is a method of treating cancer in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositionprovided herein.

In one embodiment, the cancer is colon cancer, non-small lung cancer,chronic myeloid leukemia, acute myeloid leukemia, small cell lungcancer, cervical cancer, stomach cancer, breast cancer, prostate cancer,skin cancer, melanoma, lymphoma, brain tumor, sarcoma, testicle cancer,ovarian cancer, renal cancer, head and neck cancer, liver cancer,leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia,hairy cell leukemia, T-cell prolymphocytic leukemia, large granularlymphocytic leukemia, adult T-cell leukemia, and a blood cancer.

In one embodiment, the cancer comprises a solid tumor.

A compound useful in this disclosure is administered to an appropriatesubject in need of these compounds in a therapeutically effective doseby a medically acceptable route of administration such as orally,parentally (e.g., intramuscularly, intravenously, subcutaneously,interperitoneally), transdermally, rectally, by inhalation and the like.

The term cancer is to be considered in the broadest general definitionas a malignant neoplasm, an abnormal mass of tissue, the growth of whichexceeds and is uncoordinated with that of normal tissues and persists inthe same excessive manner after cessation of the stimuli that evoked thechange. It might be added that the abnormal mass is purposeless, preyson the host, and is virtually autonomous. A cancer can also beconsidered as a malignant tumor. A further discussion of neoplasia isfound at “Robbins Pathologic Basis of Disease,” Sixth Edition, by R. S.Cotran, V. Kumar, and T. Collins, Chapter 8 (W.B. Saunders Company).This information from Chapter 8 is incorporated herein by reference. Thefollowing Table B provides examples of the types of cancers, i.e.,malignant tumors or neoplasia that may be treated by administering acompound of this disclosure.

TABLE B Tissue of Origin Malignant Composed of One Parenchymal Cell TypeMesenchymal tumors Connective tissue and derivatives FibrosarcomaLiposarcoma Chondrosarcome Osteogenic sarcoma Endothelial and relatedtissues Blood vessels Angiosarcoma Lymph vessels LymphangiosarcomaSynovium Synovial sarcoma Mesothelium Mesothelioma Brain coveringsInvasive meningioma Blood cells and related cells Hematopoietic cellsLeukemias Lymphoid tissue Malignant lymphomas Muscle SmoothLeiomyosarcoma Straited Rhabdomyosarcoma Epthelial tumors Stratifiedsquamous Squamous cell or epidermoid carcinoma Basal cells of skin oradnexa Basal cell carcinoma Epithelial lining Glands or ductsAdenocarcinoma Papillary carcinoma Cystadenocarcinoma Respiratorypassages Bronchogenic carcinoma Bronchial adenoma (carcinoid)Neuroectoderm Malignant melanoma Renal epithelium Renal cell carcinomaLiver cells Hepatocellular carcinoma Urinary tract epithelium(transitional) Transitional cell carcinoma Placental epithelium(trophoblast) Choriocarcinoma Testicular epithelium (germ cells)Seminoma Embryonal carcinoma More Than One Neoplastic Cell - MixedTumors, Usually Derived From One Germ Layer Salivary glands Malignantmixed tumor of salivary gland origin Breast Malignant cystosarcomaphyllodes Renal anlage Wilms tumor More Than One Neoplastic Cell TypeDerived From More Than One Germ Layer - Teratogenous Totipotential cellsin gonads or in Immature teratoma, embryonic rests teratocarcinoma

The compounds of the disclosure are thus useful in the treatment ofleukemia and solid tumors, such as colon, colo-rectal, ovarian, mammary,prostate, lung, kidney and also melanoma tumors. The dosage rangeadopted will depend on the route of administration and on the age,weight and condition of the patient being treated. The compounds may beadministered, for example, by the parenteral route, for example,intramuscularly, intravenously or by bolus infusion.

As used herein, a “therapeutically effective amount” of podophyllotoxinderivatives of the present invention is intended to mean that amount ofthe compound which will inhibit the growth of, or retard cancer, or killmalignant cells, and cause the regression and palliation of malignanttumors, i.e., reduce the volume or size of such tumors or eliminate thetumor entirely.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. The interrelationship ofdosages varies for animals of various sizes and species, and for humans(based on mg/m² of body surface) is described by E. J. Freireich et al.,Cancer Chemother. Rep., 50(4):219 (1966). Body surface area may beapproximately determined from the height and weight of an individual(see, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp.537-538 (1970)). A suitable dose range is from 1 to 1000 mg ofequivalent per m² body surface area of a compound of the invention, forinstance from 50 to 500 mg/m².

For all of the administering routes, the exact timing of administrationof the dosages can be varied to achieve optimal results. Generally, ifusing Intralipid 20 as the carrier for the derivative, the actual dosageof derivative reaching the patient will be less. This is due to someloss of the derivative on the walls of the syringes, needles andpreparation vessels, which is prevalent with the Intralipid 20suspension. When a carrier, such as cottonseed oil is used, this abovedescribed loss is not so prevalent because the derivative does notadhere as much to the surface of syringes, etc.

Another important feature of the method provided by the presentdisclosure relates to the relatively low apparent overall toxicity ofthe derivatives administered in accordance with the teachings herein.Overall toxicity can be judged using various criteria. For example, lossof body weight in a subject over 10% of the initially recorded bodyweight (i.e., before treatment) can be considered as one sign oftoxicity. In addition, loss of overall mobility and activity and signsof diarrhea or cystitis in a subject can also be interpreted as evidenceof toxicity.

EXAMPLES Example 1: Synthesis of N2

4-carboxyphenoxy acetic acid (2). To a suspension of 4-hydroxybenzoicacid (50 g, 361.4 mmol) and K₂CO₃ (200 g, 1.45 mol) in acetone (720 mL)was added ethyl bromoacetate (140 mL, 1.27 mol). After stirring at roomtemperature for 24 hr, the mixture was filtered and concentrated (60torr, 40° C. bath temperature). The remaining thick oil was dissolved inMeOH (720 mL) and added a solution of 30% wt/v NaOH in Water (108 g NaOHin 360 mL H₂O). The mixture was refluxed at 95° C. for 17 hr. Themixture was cooled to 0° C. and concentrated HCl (350 mL) was addedslowly while stirring. Once the addition was completed, the mixture wasfiltered and the precipitate was washed with water (1×100 mL) andacetone (2×100 mL), followed by drying under high vacuum at 40° C. for 4days to afford 42.5 g of product as white solid (60%). ESIMS: calcd forC₉H₈O₅ [M+H]⁺ 197.04, found 197.1.

Camptothecin-10-O-[4-piperidinopiperidine]carbamate (4)

The 4-piperidinopiperidinecarbamyl chloride reagent was preparedaccording to the literature condition (U.S. Pat. No. 6,121,451. Briefly,To a solution of 4-piperidinopiperidine (35 g, 208 mmol) in DCM (2.6 L)at 0° C. was added a solution of triphosgene (22.1 g, 83.2 mmol) in DCM(300 mL) slowly. The mixture was stirred from 0° C. to rt for 17 hr.Celite was added and the solution was filtered. The filtrate was washedwith 7% wt/v NaHCO₃ solution and dried over MgSO₄ (100 g). The mixturewas filtered and the solvent evaporated. The remaining residue wasdissolved in anhydrous DCM (208 mL) to make IM solution. The solutionwas used as is without further purification.

The 10-hydroxycamptothecin (25 g, 68.5 mmol) was dissolved in 1:1DCM/pyridine (1.37 L) and added the freshly prepared4-piperidinopiperidinecarbamyl chloride solution (208 mL). Afterstirring at rt for 4 hr, the reaction mixture was concentrated andpurified by silica gel chromatography (600 g) with 10% MeOH/DCM (7 L) togive 38 g of product as light yellow solid (100%). ESIMS: calcd forC₃₁H₃₄N₄O₆ [M+H]⁺ 559.25, found 559.0.

Camptothecin-10-O-[4-piperidinopiperidine]carbamyl]-20-O-[4-carboxyphenoxy]aceticacid. (5)

Camptothecin-10-O-[4-piperidinopiperidine]carbamate (4, 38 g, 68.5mmol), EDC (61.4 g, 319.6 mmol), DMAP (4.85 g, 79.9 mmol) and4-carboxyphenoxy acetic acid (2, 39.2 g, 199.7 mmol) were dissolved in1:1 DCM/DMF (2.66 L). After stirring at rt for 22 hr, the mixture wasconcentrated and the remaining residue was dissolved in DCM (3 L) andwashed with saturated NaHCO₃ solution (2×200 mL) followed by water(2×200 mL). The aqueous phase was combined and extracted with DCM (2×200mL). The organic layer and the organic extracts were combined, driedover MgSO₄ (100 g), filtered and the solvent evaporated. The remainingresidue was purified by silica gel chromatography (1 kg) with 10%MeOH/DCM+1% AcOH (2 L), 20% MeOH/DCM+2% AcOH (5 L), and 30% MeOH/DCM+3%AcOH (4 L). The product containing fractions were combined and thesolvent evaporated. The crude product was dissolved in 10% MeOH/DCM (100mL) and the solution was added to MeOH (500 mL). The mixture was stirredat rt for 30 min and the resulting precipitate was collected byfiltration to give 22.4 g of product as light yellow solid (45%). ESIMS:calcd for C₄₀H₄₀N₄O₁₀ [M+H]⁺ 737.27, found 737.0. Note: this step can beimproved further and we should be able to get ˜70% yield.

N2

EDC (17.53 g, 91.3 mmol), DMAP (3.71 g, 30.4 mmol), 5 (22.4 g, 30.4mmol) and podophyllotoxin (25.2 g, 60.9 mmol) were dissolved in 1:1DCM/DMF (1.52 L) and the mixture was stirred at rt for 24 hr. Thereaction mixture was diluted with DCM (2 L), washed with saturatedNaHCO₃ solution (2×500 mL), water (lx 500 mL), dried over MgSO₄ (100 g),filtered and the solvent evaporated. The remaining residue was purifiedby Silica gel column chromatography (900 g) with 5% MeOH/DCM (2 L) and7.5% MeOH/DCM (6 L). The product containing fractions were collected andthe solvent was evaporated. The pure product was acidified by dissolvingin 10% MeOH/DCM (50 mL) followed by addition of 4M HCl in dioxane (25mL). The mixture was added slowly to stirred Et₂O (750 mL). Theprecipitate was filtered and dried under high vacuum overnight.Recovered 17.5 g of N2 hydrochloride salt as yellow solid (50%). ESIMS:calcd for C₆₂H₆₀N₄O₁₇ [M+H]⁺ 1133.40, found 1133.0.

The ¹H NMR and ¹³C NMR spectra of N2 are shown in FIGS. 1A and 2respectively. The LC-MS spectrum of N2 is shown in FIG. 3A.

Example 2: Synthesis of N2.HCl Formulate

The N2.HCl formulate was prepared by the process shown in Scheme 5. To afiltered solution of N2 free base (1 equiv) in dichloromethane (ACS) at10±5° C. was added HCl in ether (ACS, 1 M, 1.3 equiv). The solvent wasremoved in vacuo. To the residue was added a solution of MPEG-2000-DSPE(cGMP, 2 equiv) in dichloromethane. The mixture was agitated to obtain aclear solution and then the solvent was removed in vacuo. The residuewas dissolved in dichloromethane, concentrated to dryness, dried undervacuum at room temperature until dichlormethane NMT 600 ppm by GC,micronized with a blender and screened with a 0.35 mm sieve to affordN2.HCl Formulate, lot #011000, 2.45 kg.

Example 3: Preparation of N2-HCl for Injection

L-Lactic acid solution (0.01 M, pH 3.5) was prepared by combiningsterile water for injection (SWI, 18±0.5 kg) and L-lactic acid (18.02g), adding 0.5 N sodium hydroxide until the pH of the solution is3.5±0.1 and adding sufficient SWI to bring the total quantity ofsolution to 20.00 kg. The pH was adjusted to 3.5±0.1 by addition of 0.5N sodium hydroxide.

A mixture of N2.HCl Formulate (697.7 g) and 0.01 M L-lactic acidsolution, pH 3.5 (17±0.2 kg) was stirred until a solution was obtained.d-Mannitol (168.0 g) was added with mixing. The pH of the resultingsolution was adjusted to 3.5±0.1 by addition of 0.5 N sodium hydroxide.Sufficient 0.01 M L-lactic acid solution, pH 3.5 was added to bring thetotal quantity of solution to 20.00 kg. The pH was adjusted to 3.5±0.1by addition of 0.5 N sodium hydroxide. The solution was filtered througha 0.22 micron sterile filter into a sterile carboy and then filled intosterile 10 mL vials with 5.00±0.10 g per vial. Lyophilization wasperformed under sterile conditions ending at +25° C. and 100 mTorr. Thevacuum was broken with nitrogen NF and sterile seals applied to thevials, which were stored at 2-8° C.

Example 4: Synthesis of Target Compound 1

Target Compound 1 (also referred to herein as N1) can be synthesizedfollowing the procedures described above for N2 with any necessarymodifications that will be apparent to a skilled artisan (see Scheme 6).The ¹H NMR spectrum and LC-MS spectrum of Target Compound 1 are shown inFIGS. 1B and 3B respectively.

Example 5: Synthesis of Target Compound 3

Target Compound 3 (also referred to herein as N3) can be synthesizedfollowing the procedures described above for N2 with any necessarymodifications that will be apparent to a skilled artisan (see Scheme 7).The ¹H NMR spectrum and LC-MS spectrum of Target Compound 3 are shown inFIGS. 1C-1E and 3C respectively.

Example 6: Synthesis of Target Compound 4

Target Compound 4 (also referred to herein as N4) can be synthesizedfollowing the procedures described above for N2 with any necessarymodifications that will be apparent to a skilled artisan (see Scheme 8).The ¹H NMR spectrum and LC-MS spectrum of Target Compound 4 are shown inFIGS. 1F and 3D respectively.

1.1. Background on Biological Assays

Studies were conducted using N2 and Target Compounds 1, 3, and 4. Invitro efficacy studies demonstrated that these compounds were veryeffective in killing human colon and lung cancer cells, and especiallyresistant colon cancer cells (overexpressing multi-drug resistant gene 1[MDR1]). To further evaluate its preclinical efficacy, the in vivoefficacy studies have been conducted on two colon cancer models and onefor Chronic Myeloid Leukemia (CML) model. The toxicity of four leadcompounds were remarkably low in mice. Among them, N2 (also referred toherein as 801) is a promising compound with lower toxicity and higherefficacy against human cancer xenografts. The results showed that801-HCl and formulated 801 are both effective for the human colon cancerand CML xenograft models.

1.2 In Vitro Efficacy Studies of Podophyllotoxin Analogs

1.2.1. Colony Formation Assay

Cancer cells were plated in 60 mm Petri dishes containing 2.7 ml ofmedium (modified McCoy's 5a medium containing 10% fetal bovine serum and100 units/ml penicillin and 100 ug/ml streptomycin). The cells wereincubated in a CO₂ incubator at 37 degree C. for 5 hours for attachmentto the bottom of Petri dishes. Drugs were made up fresh in medium at tentimes the final concentration, and then 0.3 ml of this stock solutionwas added to the 2.7 ml of medium in the dish. The cells were thenincubated with drugs for 72 hours at 37 degree C. At the end ofincubation the drug-containing media were decanted, the dishes wererinsed with 4 ml of Hank's Balance Salt Solution (HBSS), 5 ml of freshmedium was added, and the dishes were returned to the incubator forcolony formation. The cell colonies were counted using colony counterafter incubation for 7 days (HCT116 cell line), 14 days (VM46 cell line)and 15 days (H23 cell line). Cell survival (%) was calculated from 3repeated dishes for each drug concentration and the mean values of IC50(the drug concentration producing 50% inhibition of colony formation)were determined for each tested compound.

1.2.2. Chemotherapeutic Effects on HCT116 Human Colon Cancer Cells

Chemotherapeutic Effects of podophyllotoxin Compounds (N1-4) on HCT116Human Colon Cancer Cells are tabulated below.

Compound IC₅₀ (HCT116) N1 24.2 nM N2 (801) above 500 nM N3 282.7 nM N46.6 nM1.2.3. Chemotherapeutic Effects on VM46 Human Colon Cancer CellsChemotherapeutic Effects of podophyllotoxin Compounds (N1-4) on VM46Human Colon Cancer Cells Overexpressing Multi-drug Resistant Gene 1 aretabulated below.

Compound IC₅₀ (VM46) N1 8 nM N2 (801) approximate 520 nM N3 300 nM N4 5nM1.2.4. Chemotherapeutic Effects on H23 Human Non-Small Lung Cancer CellsChemotherapeutic Effects of podophyllotoxin Compounds (N1-4) on H23Human Non-small Cell Lung Cancer Cells are tabulated below.

Compound IC₅₀ (H23) N1 7.05 nM N2 (801) 461.76 nM N3 200 nM N4 12.94 nM

In summary, N1, N2(801), N3, N4 all showed effectiveness against the 3selected cell lines at nM level concentrations. N1 and N4 showed goodeffectiveness at very low concentration. Among the 3 cell lines, VM46Human Colon Cancer Cells overexpress Multi-drug Resistant Gene 1 (MDR1).All compounds overcame MDR1 mediated drug resistance. N2(801)demonstrated more effectiveness against non-small cell lung cancer thancolon cancer.

1.3. Toxicity Studies of Compounds N1-4 in Normal Mice

1.3.1. Method:

In the toxicity studies, 2 C3H female normal mice were dosed withcompounds N1-4 (N1, N2, N3, and N4) at low and moderate doses of 40 and100 mg/kg. Since no severe and irreversible toxicity occurred at thesedoses, a new pair of animals was initiated at 120 mg/kg which was 1.2times higher than 100 mg/kg. Sequential dosages (ie. 2 mice for eachdrug dose) were increased by a factor of 1.2-1.5 until irreversibletoxicity was observed (animal became emaciated; animal underwent rapidweight loss (cachexia) (20%) within several (2-4) days)(euthanasia wasrequired) occurred. Then another pair of animals was initiated at thehighest nontoxic dosage, and successive dosages were increased by afactor of 1.15. The result of this exercise were two dosages, one thatwas apparently nontoxic and the other that demonstrated irreversibletoxicity that required euthanasia, separated by a factor of 1.15. Sixmice were dosed at each dosage. If no irreversible toxicity occurred atthe lower dosage and at least one with irreversible toxicity at thehigher dose, then the lower dose was considered to be the MTD. HCL saltsof N1-4 were formulated in a solution of ethanol:Cremophor EL:0.2MNaAc:saline (5:5:10:80) with a final pH of 5.0-5.5. N1-4 bases wereformulated in a solution of ethanol: Cremophor EL:saline (5:5:90). Allcompounds were administered to C3H female normal mice by a single i.p.injection at a volume of 0.5 ml/20 g mouse. Drug toxicity was evaluatedon mice checked daily for 40 days. The toxicity parameters reported werethe MTD₄₀. The MTD was defined as the highest dose causing noirreversible toxicity in one treatment group, but at least one animalexhibiting irreversible toxicity and being euthanized at next higherdose. At the end of experiment, all mice were euthanized.

1.3.2. Results:

The evaluation of the toxic effects of N1, N2, N3, and N4 in C3H femalenormal mice have been completed. As shown in Table 1.3.1, the MTD valuesof all the compounds were significantly higher than many clinicallyavailable anticancer drugs. The higher its MTD, the lower its toxicityto normal mice. Among the four compounds, N2 was least toxic to normalmice. It is noted that the MTD of its base form was equal to that of itsHCL salt form. The next less toxic one was Neovia4. Its base form wasless toxic to mice than its HCL salt form, suggesting the lesssolubility of its base form might contribute to this difference.

TABLE 1.3.1 The MTD Values of Compounds N1-4 in C3H mice. Names ofCompounds MTD Values (mg/kg) N1 HCL Salt 120 N2 Base 300 N2 HCL Salt 300N3 HCL Salt 144 N4 Base 250 N4 HCL Salt 1801.4. Preliminary In Vivo Efficacy Study of Four Podophyllotoxin Analogsfor Selection of the Most Promising Candidate Drug

To evaluate in vivo efficacy of four compounds N1-4 on HCT116 humancolon cancer, in vivo anticancer efficacy study was performed on nudemice (nu/nu genotype) bearing HCT116 human colon cancer xenografts.

1.4.1. Method

In vivo anticancer efficacy study was performed on nude mice (nu/nugenotype) bearing HCT116 human colon cancer xenografts. These humantumors grew exponentially following implantation into the flanks of themice and reached an average tumor volume of 79.85 cu. mm. Treatment wasinitiated at that time, with the first day of treatment designated asday 0 for all calculations and plots. The 6 mice were injected i.p. witheach of four Neovia drug solutions (5% alcohol, 5% Cremophor EL, and 90%saline, 0.5 ml/20 g, single injection). Drug doses (i.p. MTD doses) were120 mg/kg for Neovia-1, 300 mg/kg for Neovia-2, 120 mg/kg for Neovia-3,and 180 mg/kg for Neovia-4. Control group of mice was treated withvehicle alone. After treatment, tumor sizes were measured by caliperevery other day. The measurement of the tumor diameters (d1, d2) in twoorthogonal directions was used to calculate the tumor volume (tumorvolume=π/6 {(d1+d2)/2}3) using the approximation that the tumors arespherical. Each tumor growth curve for each mouse was plotted. The time(days) required to grow the tumor to the size of 800 cu. mm aftervarious treatments was calculated as tumor growth delay indexes forevaluating the treatment effects. Survival times after treatment wasalso observed and recorded.

1.4.2. Results

All four compounds N1-4 after a single i.p. injection proved to behighly effective in inhibiting the growth of HCT116 human colon cancerxenografts. The results showed that N2 (801) would be the most promisingcandidate drug against HCT116 human colon cancer among all 4 compounds.

1.5. In Vivo Efficacy Study: 801 in Treatment of Subcutaneous COLO205Human Colon Cancer Xenograft Model.

1.5.1. Objective

To evaluate preclinically the in vivo therapeutic efficacy and safety ofcompound 801 in the treatment of the subcutaneous COLO205 human coloncancer xenograft model and compared with the SOC drug irinotecan.

1.5.2. Animals

Descriptor Type Species Mus Musculus Strain BALB/c nude Age 6-8 weeksSex Female Body weight 18-22 g Number of animals 40 mice Animal supplierBeijing Vital River Inc1.5.3. Method

Each mouse was inoculated subcutaneously with 5×10⁶ COLO205 tumor cellsin 0.1 ml PBS for tumor development. Treatments were started when thetumor volume reached 120 mm³. The major endpoint was to see if the tumorgrowth could be delayed or regressed. Body weight and tumor volume weremeasured twice a week. Tumor volume was then used for calculations ofboth T-C and tumor growth inhibition (TGI) values.

1.5.4. Groups and Treatments

Treatment Number of Dosage Route/ Dosing Number of Group (Vehicle)animals (mg/kg) schedule Volume Injections 1 Vehicle: 8 — i.v., q5d x 346.81 μl/g 4 injections, mPEG-2000- BW 1.5 hrs interval DSPE (13.33mg/ml) 2 mPEG-2000- 8 125 i.v., q5d x 3 46.81 μl/g 4 injections, DSPE BW1.5 hrs interval formulated 801 3 mPEG-2000- 8 100 i.v., q3d x 3 37.45μl/g 3 injections, DSPE BW 1. hrs interval formulated 801 4 801-HCI 8125 i.p., q3d x 3 10 μl/g BW, one bolus 5 Irinotecan 8 70 i.p., q3d x 31.5.5. Statistics

The differences between the mean values of tumor volume for comparinggroups were analyzed for significance using the one-way ANOVA test.P<0.05 was considered as statistically significant.

1.5.6. Results

The results from show i.v. mPEG-2000-DSPE formulated 801 at dose levelsof 125 mg/kg (q5d×3) and 100 mg/kg (q3d×3) produced a moderate antitumoractivity with TGI values of 37% and 42%, respectively (p=0.008 andp=0.002 vs. control).

The results show i.p. 801-HCl at 125 mg/kg and irinotecan at 70 mg/kgproduced a strong antitumor activity with TGI values of 70% and 97% atday 29 (both p<0.001 vs. control).

FIG. 4A shows the antitumor activity of formulated N2-HCl (i.v.) intreatment of COLO205 Human Colon Cancer Xenograft Model and FIG. 4Bshows the antitumor activity of formulated N2-HCl (i.p.) in treatment ofCOLO205 Human Colon Cancer Xenograft Model.

Regarding the safety profile, the mice tolerated the treatments well; nosevere body weight loss or other gross clinical abnormalities wereobserved during the treatment period.

1.5.7. Conclusion

801-HCl at 125 mg/kg (i.p. delivery) demonstrated a strong antitumoractivity with a good safety profile. mPEG-2000-DSPE formulated 801 (i.v.delivery) produced a moderate efficacy, which might be caused either bya splited injections for each i.v. dosing due to a limitation in dosingvolume allowance for i.v. injection in mice.

1.6. In-Vivo Efficacy Study: 801 in Treatment of Subcutaneous HCT116Human Colon Cancer Xenograft Model

1.6.1. Objective

To evaluate preclinically the in vivo therapeutic efficacy and safety ofcompound 801 in the treatment of the subcutaneous HCT116 human coloncancer xenograft model in nude mice.

1.6.2. Animals

Descriptor Type Species Mus Musculus Strain BALB/c nude Age 6-8 weeksSex Female Body weight 18-22 g Number of animals 40 mice Animal supplierBeijing Vital River Inc1.6.3. Method

Each mouse was inoculated subcutaneously with 5×10⁶ HCT116 tumor cellsin 0.1 ml PBS for tumor development. Treatments were started when thetumor volume reached 100 mm³. The major endpoint was to see if the tumorgrowth could be delayed or regressed. Body weight and tumor volume weremeasured twice a week. Tumor volume was then used for calculations ofboth T-C and tumor growth inhibition (TGI) values.

1.6.4. Groups and Treatments

Treatment Number of Dosage Route/ Dosing Number of Group (Vehicle)animals (mg/kg) schedule Volume Injections 1 Vehicle: 8 — i.v., q5d x 346.81 μl/g 4 injections, mPEG-2000- BW 1.5 hrs interval DSPE (13.33mg/ml) 2 mPEG-2000- 8 125 i.v., q5d x 3 46.81 μl/g 4 injections, DSPE BW1.5 hrs interval formulated 801 3 mPEG-2000- 8 100 i.v., q3d x 3 37.45μl/g 3 injections, DSPE BW 1. hrs interval formulated 801 4 801-HCI 8125 i.p., q3d x 3 10 μl/g BW, one bolus 5 Irinotecan 8 70 i.p., q3d x 31.6.5. Statistics

The differences between the mean values of tumor volume for comparinggroups were analyzed for significance using the one-way ANOVA test.P<0.05 was considered as statistically significant.

1.6.6. Results

The results from FIG. 5A show i.v. mPEG-2000-DSPE formulated 801 at doselevels of 125 mg/kg (q5d×3) and 100 mg/kg (q3d×3) produced a moderateantitumor activity with TGI values of 52% and 49%, respectively (p=0.002and p=0.001 vs. control).

The results from FIG. 5B show i.p. 801-HCl at 125 mg/kg and irinotecanat 70 mg/kg produced a similar and strong antitumor activity with TGIvalues of 93% and 92% at day 29 (both p<0.001 vs. control).

FIG. 5A shows the antitumor activity of formulated N2 (i.v.) intreatment of HCT116 Human Colon Cancer Xenograft Model. FIG. 5B showsthe antitumor activity of N2-HCl (i.p.) in treatment of HCT116 HumanColon Cancer Xenograft Model.

Regarding the safety profile, a minor body weight loss (4-6%) wasobserved for 801-HCl and formulated 801 treated animals, whileirinotecan resulted in 8% body weight loss. No other gross clinicalabnormalities were observed during the treatment period.

1.6.7. Conclusion

801-HCl at 125 mg/kg (i.p.) demonstrated a strong antitumor activitywith a good safety profile. mPEG-2000-DSPE formulated 801 (i.v.)produced a moderate efficacy, which might be caused either by splitedinjections for daily iv dosing due to a limitation in volume allowancefor iv injection in mice.

1.7. In Vivo Efficacy Study: 801 in Treatment of Subcutaneous K562 HumanChronic Myeloid Leukemia Xenograft Model.

1.7.1. Objectives

Preclinically evaluate in vivo therapeutic efficacy and safety ofcompound 801 in the treatment of the subcutaneous human chronic myeloidleukemia xenograft model in NOD/SCID mice.

1.7.2. Animals

Descriptor Type Species Mus Musculus Strain NOD/SCID Age 6-8 weeks SexFemale Body weight 22-26 g Number of animals 32 mice Animal supplierBeijing HFK Bio-Technology Co., Ltd.1.7.3. Method

The mice were irradiated (2Gy) using a Co60 irradiator source. After 24hours, each animal was inoculated subcutaneously on the right flank withK562 cells (1×10⁷/animal) in 0.1 mL of PBS for tumor development. Tumordevelopment was allowed undisrupted until the mean volume reachedapproximately 150 mm³. The major endpoint was to see if the tumor growthcould be delayed or regressed. Body weight and tumor volume weremeasured. Tumor volume was then used for calculations of both T-C andtumor growth inhibition (TGI) values.

1.7.4. Groups and Treatments

TABLE 1.7 Number of Dose Route of Days of Dose Group animals Treatment(mg/kg) administration Treatment Volume 1 8 Vehicle: same as iv TID onmPEG-2000- Group 2. Days 8, 11, DSPE 14 2 8 mPEG-2000- 60 iv TID on 20μl/g DSPE Day 8 formulated 801 3 8 801-HCl 300 ip Days 8, 11, 20 μl/g 144 8 Imatinib 75 po Days 8-21 10 μl/g. mesylate1.7.5. Statistics

The differences between the mean tumor volumes of comparing groups wereanalyzed for statistical significance using one-way ANOVA test. P<0.05was considered as statistically significant.

1.7.6. Results

The control animals were euthanized on Day 25 for large tumor burden.

The mPEG-2000-DSPE vehicle was tolerated well by the control animals.However, the mPEG-2000-DSPE formulated 801 at the test dosage of 60mg/kg (iv, TID dosing) caused severe toxicity to the animals and onlyone TID dosing was delivered; a drastic BW loss of 14.5% was observed onthe next day after the Day 8 dosing, with 2 out of 8 animals died onDays 9 and 10. The test agent 801-HCl was tolerated well by the animalswhen delivered ip at a dosage of at 300 mg/kg, with only a maximum 7.7%BW loss recorded on the next day after the 2nd dosing. The positive drugimatinib was tolerated well by the animals, and a minor BW loss of 6.2%was observed at the end of the treatment.

The mean TVs of different groups are presented in Table 1.7.1. The TGIvalues on Day 25 are shown in Table 1.7.2. The tumor growth curves ofthe different mice groups tabulated in Table 1.7 are presented in FIG.6.

The mean TV of control animals reached 3,467 mm³ on Day 25 after thetumor cell inoculation. Test agent mPEG-2000-DSPE formulated 801 (60mg/kg, iv, TID: total 180 mg/kg), administered for only one-day due totolerability issue, still produced moderate antitumor activity with aTGI value of 55% (p=0.001 vs. control). Test agent 801-HCl at 300 mg/kg(ip) exhibited a drastic antitumor activity with a TGI value of 98%(p<0.001 vs. control). For this treatment group, 3 out 8 animalsachieved complete remission of the tumors by Day 21 (7 days after thelast dosing) without further tumor regrowth by the end of the study. Incomparison, imatinib at 75 mg/kg(po) exhibited only minor antitumoractivity with a TGI value of 33%, which is not significant from thecontrol (p=0.064 vs. control).

TABLE 1.7.1 Mean Tumor Volumes. Mean Tumor Volume (mm³) mPEG-2000- DSPEImatinib Vehicle formulated 801 801-HCl mesylate mPEG-2000- 60 mg/kg,TID 300 mg/kg, 75 mg/kg, Days¹ DSPE on Day 8 Days 8, 11, 14 Days 8-21  8156 ± 12 159 ± 9  159 ± 16  156 ± 12 11 403 ± 52 276 ± 23 221 ± 27  324± 57 14 851 ± 83 300 ± 29 164 ± 16   545 ± 105 18 1,778 ± 206  576 ± 9747 ± 7 1,146 ± 152 21 2,259 ± 150   933 ± 178 39 ± 6 1,596 ± 210  25²3,467 ± 270  1,581 ± 245   74 ± 12 2,368 ± 268 28 — 2,032 ± 231   95 ±19 2,761 ± 283 Values are presented as mean ± standard error. ¹Daysafter tumor inoculation. ²The control group was terminated on Day 25 forlarge tumor burden.

TABLE 1.7.2 Antitumor Activity of 801 in Treatment of the SubcutaneousK562 Human Chronic Myeloid Leukemia Model. Mean TVs P values¹ (mm³,Dunnett T3 TGI CR Groups D25) test (Day 25²) ratio³ G1: Vehicle 3,467 ±270 — — — mPEG-2000-DSPE G2: mPEG-2000-DSPE 1,581 ± 245 0.001 55 —formulated 801 G3: 801-HCl   74 ± 12 <0.001 98 3/8 G4: Imatinib mesylate2,368 ± 268 0.064 33 — TGI = Tumor growth inhibition. ¹vs. G1: Vehicleon Day 25. ²Days after tumor inoculation. ³The ratio of completeregression. Other comparisons: G2 vs. G3, p = 0.007; G2 vs. G4, p =0.244; G3 vs. G4, p < 0.001.1.7.7. Conclusion

801-HCl at 300 mg/kg (i.p.) exhibited a drastic antitumor activity witha good safety profile. mPEG-2000-DSPE formulated 801 (60 mg/kg, iv, TID:total 180 mg/kg), administered for only one-day due to tolerability inthe mice irradiated at 2Gy using a Co60 irradiator source, stillproduced moderate antitumor activity.

1.8. In Vivo Efficacy Study: 801 in Treatment of K562 Human ChronicMyeloid Leukemia Xenograft Model (2^(nd) K562 Study).

1.8.1. Objectives

The objective of this study was to evaluate preclinically in vivotherapeutic efficacy and tolerability of the test article, compound 801,in treatment of the subcutaneous K562 human chronic myeloid leukemiaxenograft in NOD SCID mice, and compared with these of SOC drugimatinib.

1.8.2. Animals

Descriptor Type Species Mus Musculus Strain NOD/SCID Age 6-8 weeks SexFemale Body weight 18-22 g Number of animals 48 mice plus spare Animalsupplier Beijing Vital River Inc., Beijing, China1.8.3. Method

The mice were irradiated (200 rad) using a Co60 irradiator source. After24 hours, each animal was inoculated subcutaneously on the right flankwith K562 cells (1×107/animal) in 0.1 mL of PBS for tumor development.Tumor development was allowed undisrupted until the mean volume reachedapproximately 130 mm3. Animals were then randomized into 6 groups, witheach group consisting of 8 animals. The test articles were administeredto the tumor-bearing animals according to predetermined regimens asshown in the experiment design table above.

1.8.4. Groups and Treatments

TABLE 1.8 Dose (mg/ Group n Treatment kg) ROA schedule 1 8 Vehicle: —i.v. q3d × 2 (bid injection) mPEG-2000-DSPE q3d × 4 (qd injection) 2 8Imatinib 75 p.o. qd × 14 3 8 mPEG-2000-DSPE 150 i.v. q3d × 2 (tidinjection) formulated 801 4 8 mPEG-2000-DSPE 100 i.v. q3d × 2 (bidinjection) formulated 801 5 8 mPEG-2000-DSPE 50 i.v. q3d × 8 (qdinjection) formulated 801 6 8 mPEG-2000-DSPE 25 i.v. q3d × 8 (qdinjection) formulated 801 n = number of animals; ROA = route ofadministration; i.v. = intravenously; p.o. = per os (orally). Dosingvolume for formulated 801 and the vehicle control was 20 μl/g based onbody weight, the dosing volume for Imatinib solution was 10 μl/g.1.8.5. Statistics

The differences between the mean tumor volumes of comparing groups wereanalyzed for statistical significance using one-way ANOVA test. P<0.05was considered as statistically significant.

1.8.6. Results

a. 1.8.6.1 Tumor Volumes

The tumor sizes of the different groups are shown in Table 1.8.1.

TABLE 1.8.1 Tumor Sizes in the Treatment Groups Mean Tumor Volume (mm³)¹Vehicle: Imatinib Formulated Formulated Formulated Formulated mPEG-2000-mesylate 801 801 801 801 DSPE 75 mg/kg 150 mg/kg 100 mg/kg 50 mg/kg 25mg/kg Days² i.v. q3d x 6 p.o. qd x 14 i.v. q3d x 2 i.v. q3d x 2 i.v. q3dx 8 i.v. q3d x 8  8  130 ± 9  129 ± 10 129 ± 10 128 ± 7  127 ± 9  129 ±7  11  324 ± 29  206 ± 32 135 ± 14 138 ± 16 185 ± 16 192 ± 15 14  607 ±71  423 ± 75 163 ± 15 221 ± 29 260 ± 29 354 ± 26 18 1,290 ± 81  765 ±121 338 ± 47 453 ± 58 474 ± 57 666 ± 48 21 1,643 ± 95 1,094 ± 123 639 ±81 826 ± 85 585 ± 59 936 ± 89  25³  2,401 ± 167 1,504 ± 120 1,013 ± 105 1,222 ± 107  776 ± 69 1,305 ± 147  28 — 2,058 ± 168 1,514 ± 146  1,589 ±109  1,007 ± 134  1,556 ± 180  32 — 2,676 ± 236 2,213 ± 247  2,321 ±193  1,435 ± 133  1,976 ± 216  ¹Values are presented as Mean ± SEM ²Daysafter tumor inoculation. ³The control group was terminated on Day 25 forlarge tumor burden.

b. 1.8.6.2 Tumor Growth Inhibition

The tumor growth inhibition was summarized in Table 1.8.2. The tumorgrowth curves of different mice groups treated with formulated 801 andimatinib as tabulated in Table 1.8 are presented in FIG. 7.

TABLE 1.8.2 Antitumor Activity of formulated 801 and Imatinib in theTreatment of the K562 Human Chronic Myeloid Leukemia Xenograft ModelTumor Size TGI Treatment (D25, mm³)¹ (%) P value² G1: Vehicle Control2,401 ± 167 — — G2: Imatinib mesylate 75 mg/kg 1,504 ± 120 37 <0.001 G3:Formulated 801 150 mg/kg 1,013 ± 105 62 <0.001 G4: Formulated 801 100mg/kg 1,222 ± 107 51 <0.001 G5: Formulated 801 50 mg/kg  776 ± 69 70<0.001 G6: Formulated 801 25 mg/kg 1,305 ± 147 45 <0.001 ¹Values arepresented as Mean ± SEM ²vs. vehicle control; P values: G2 vs G3 =0.013; G2 vs G4 = 0.144; G2 vs G5 <0.001; G2 vs G6 = 0.262; G3 vs G4 =0.305; G3 vs G5 = 0.248; G3 vs G6 = 0.130; G4 vs G5 = 0.033; G4 vs G6 =0.665; G5 vs G6 = 0.008;1.8.7 Conclusion

Formulated 801 at the dose of 50 mg/kg with q3d×8 treatment scheduledemonstrated a strong antitumor activity in K562 human chronic myeloidleukemia xenograft model, but under the toxicity. The dose of 50 mg/kgwas significantly more effective against K562 CML xenografts than thedose of 25 mg/kg using the same treatment schedule, revealing adose-response relationship. Formulated 801 at dose level (50 mg/kg)showed significantly the stronger antitumor activity than that ofimatinib (p<0.001).

2.0 Safety Studies

The toxicity studies in normal Cynomolgus monkeys showed that afterdosing with 801 HCl via intravenous infusion twice daily with a 5-hourinterval, the MTD was the 60 mg/kg/day. The results of a 5-day repeatdose toxicity study of 801 HCl following intravenous infusion inCynomolgus Monkeys exhibited the MTD of 801 was 30 mg/kg/day forrepeating dosing.

In the rat MTD study, Sprague-Dawley rats were dosed with 801 HCl at aconcentration of 6 mg/ml via intravenous infusion or slow intravenousinjection. The MTD of 801 in normal rats was 120 mg/kg/day. In the 7-dayrepeating dose study, the rats were administered with 891 via BID slowIV injection for 7 days. The data showed that the MTD of 801 was 60mg/kg/day for repeating doing.

The invention claimed is:
 1. A compound of formula (I):

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing, wherein R¹ is H or has a structure of:

R³ is H, an optionally substituted C₁-C₈ alkyl, an optionallysubstituted 3-10 membered cycloalkyl, an optionally substituted 3-10membered heterocyclyl, an optionally substituted 6-10 membered aryl, oran optionally substituted 5-10 membered heteroaryl; R⁵ is H or has astructure of:

 and R¹¹ and R¹² independently is an optionally substituted C₁-C₈ alkyl.2. The compound of claim 1, wherein R¹ is H.
 3. The compound of claim 1,wherein R¹ has the structure of:


4. The compound of claim 1, wherein R³ is H.
 5. The compound of claim 1,wherein R⁵ is:


6. The compound of claim 1, wherein R¹¹ and R¹² independently is C₁-C₈alkyl.
 7. The compound of claim 6, wherein R¹¹ is methyl and R¹² ismethyl.
 8. The compound of claim 1, wherein R⁵ is H.
 9. The compound ofclaim 1, wherein R³ is C₁-C₈ alkyl.
 10. The compound of claim 1, whereinR³ is an ethyl.
 11. The compound of claim 1 selected from the groupconsisting of:

or an N-oxide thereof, or a pharmaceutically acceptable salt of eachthereof, or a pharmaceutically acceptable solvate of each of theforegoing.
 12. A pharmaceutical composition comprising a compound ofclaim 1 and at least one pharmaceutically acceptable excipient.
 13. Thepharmaceutical composition of claim 12, wherein the pharmaceuticalcomposition is a solid form, liquid form, injectable form, or aliposomal formulation.
 14. A pharmaceutical composition comprising acompound of claim 11 and at least one pharmaceutically acceptableexcipient.
 15. The pharmaceutical composition of claim 14, wherein thepharmaceutical composition is a solid form, liquid form, injectableform, or a liposomal formulation.